African swine fever virus impairs porcine alveolar macrophages bactericidal function by disrupting lysosomal acidification and cathepsin activity.

African swine fever virus (ASFV) is a devastating pathogen that poses a severe threat to the global swine industry. ASFV primarily targets the porcine monocyte-macrophage system, which is crucial for defending against bacterial infections via phagocytosis and subsequent intracellular degradation. Clinically, ASFV infection can be complicated by severe secondary bacterial infections. This creates a compelling paradox: while prior in vitro studies indicate that ASFV actually increases the phagocytic activity of porcine alveolar macrophages (PAMs), clinical observations frequently report severe secondary bacterial infections. This contradiction led us to hypothesize that the downstream intracellular bactericidal clearance might be compromised. Here, utilizing an in vitro co-infection model, it was demonstrated that ASFV significantly impairs the bactericidal capacity of PAMs against representative bacteria (Escherichia coli, Glaesserella parasuis, and Streptococcus suis), facilitating their intracellular survival and persistence. Although ASFV infection triggers massive reactive oxygen species (ROS) production, this oxidative stress remains functionally ineffective because phagolysosomal acidification and structural integrity are profoundly impaired. Mechanistically, at the late stage of infection, ASFV launches a multipronged assault on the endolysosomal network. Structurally, ASFV inhibits phagosomal and lysosomal acidification while inducing oxidative stress-driven severe lysosomal membrane permeabilization (LMP), leading to reduced phagolysosome volume and physical depletion of the lysosomal pool. Molecularly, ASFV impairs degradative capacity through the transcriptional suppression and blunted lysosomal enrichment of vacuolar (H+) ATPase (V-ATPase) subunits, alongside the disruption of lysosomal protease cathepsin D (CTSD) maturation. Furthermore, systematic screening identified six core candidate viral proteins, including CP530R, D129R, E183L, O174L, Q706L, and QP509R, that profoundly suppress both ATP6V0D and CTSK transcription, further exacerbating this functional impairment. Collectively, our in vitro findings reveal that ASFV dismantles host phagolysosomal acidification, thereby neutralizing the microbicidal potential of infected macrophages and potentially converting them into a permissive niche for secondary bacterial pathogens. These observations provide critical new mechanistic insights that may help explain ASFV-associated immune dysfunction at the cellular level.